Fig. 2: Tumor-directed, non-cytotoxic radiation promotes local immunosurveillance in the tumor microenvironment. | Nature Communications

Fig. 2: Tumor-directed, non-cytotoxic radiation promotes local immunosurveillance in the tumor microenvironment.

From: The tumor-sentinel lymph node immuno-migratome reveals CCR7⁺ dendritic cells drive response to sequenced immunoradiotherapy

Fig. 2

A (left) Cartoon schema of tumor-directed radiation therapy. (right) Representative CT images of sagittal, coronal, and axial series overlaid with radiation planning. Animals were anesthetized with isoflurane and positioned within the small animal radiotherapy machine. A spiral CT scan with 1 mm cuts of the neck was obtained, and cervical lymphatics were delineated as the planning target volume. A 5 mm collimator was installed, and two static parallel opposed beams were used to deliver homogenous single fraction doses to the planned target volume. Representative of n = 10 biologically independent samples; the experiment was independently repeated at least twice with similar results. B Cartoon schema of the experimental approach. WT animals were injected with 4MOSC1 and then treated with tdRT on day 6. C Representative H&E staining of tumor sections from control and tdRT-treated groups (4 Gy, 8 Gy, 12 Gy tdRT) at week 3 post-treatment. Tumor samples were fixed in zinc formalin fixative and sent for embedding, sectioning, and H&E staining. Slides were analyzed using QuPath software. Representative of n = 3 biologically independent samples; experiment was independently repeated at least twice with similar results. D Tumor growth curves for control and tdRT-treated groups (4 Gy, 8 Gy, 12 Gy tdRT) from 4MOSC1- and 4MOSC2-tumor bearing animals (top and bottom, respectively). Tumor volumes were measured over time, with significant differences observed at specific time points (n = 6 mice per group for Control, 6 mice for 4 Gy, 6 mice for 8 Gy, and 6 mice for 12 Gy, p = ns for 4 Gy, p < 0.0001 for 8 Gy and 12 Gy). p values calculated by two-sided unpaired Student’s t-test. Best-fit lines and p values calculated by simple linear regression (two-sided). E Pathway enrichment analysis of immune-related gene expression changes in tumors following tdRT. Key pathways include chemokine response, myeloid leukocyte migration, and regulation of inflammatory response. Tumors were harvested, and RNA was isolated using Qiagen RNeasy Mini Columns. Library preparation and paired-end RNA sequencing were performed by Novogene. Gene set enrichment analysis was conducted using the GSEAPreranked module on the GenePattern public server, with the Gene Ontology (Biological Processes) and ImmunesigDB gene set collections used. X-axis represents gene sets ranked by normalized enrichment score (NES); Y-axis represents the −log₁₀(FDR q-value). F (left) Cartoon schema of the experimental approach. WT animals injected with 4MOSC1 and then treated with tdRT on day 6 followed by tumor harvest on day 10. (right) Flow cytometric analysis of trafficking CXCR3 + CD8 + T cells and migratory MHCII + CCR7+ dendritic cell (DC) populations in the TME post-tdRT. Quantification is shown (n = 8–10 biologically independent samples per group, p = 0.0046 for CD8 + T cells, p = 0.0198 for DCs). Tumors were isolated, minced, and processed into single-cell suspensions using the Tumor Dissociation Kit and gentleMACS Octo Dissociator. Flow cytometry was performed using fluorochrome-conjugated antibodies. Data are presented as mean values ± SEM; p values calculated by two-sided unpaired Student’s t-test. G CD47 expression on tumor cells post-tdRT, measured by flow cytometry and shown as normalized median fluorescence intensity (MFI) on live CD45- cells (n = 9 biologically independent samples per group, p = 0.0013). Data are presented as mean values ± SEM; p values calculated by two-sided unpaired Student’s t test. H (left) Cartoon schema of the experimental approach. WT animals injected with 4MOSC1-LucOS (ovalbumin expressing) and then treated with tdRT on day 6 followed by tumor harvest on day 10. (right) Flow cytometric analysis of H-2Kb-SIINFEKL expressing antigen-presenting cells post-tdRT. Quantification of normalized populations is shown (n = 7 biologically independent samples per group, p = 0.0397). Tumors were processed as described above, and flow cytometry was performed using H-2Kb-SIINFEKL specific antibodies. Data are presented as mean values ± SEM; p values calculated by two-sided unpaired Student’s t-test. Created in BioRender. Saddawi-Konefka, R. (2025) https://BioRender.com/m675tdd. Source data are provided as a Source Data file.

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